Method for testing a cell sample

ABSTRACT

In this invention, a cell parameter, for example cell volume, is determined by subjecting one or more aliquots of a sample cell suspension to one or more alterations of at least one parameter of the cell environment to identify a point at which the cells achieve a particular shape to obtain a sample specific shape compensation factor. Preferably, the environmental parameter change is a reduction in osmolality.

TECHNICAL FIELD

[0001] This invention relates to a method for testing a cell sample or afluid sample.

[0002] Many types of cells as well as artificial cells may be testedaccording to the method of the present invention, although the test isespecially suitable for testing red and white blood cells.

BACKGROUND ART

[0003] It is well known that certain diseases give rise to changes inthe condition of blood and especially of the red blood cells. Bloodcharacteristics such as red blood cell count, mean cell volume,haemoglobin content and haematocrits are commonly used in the diagnosisof disease.

[0004] The measurement of cell volume is one of the most informativeinvestigations in clinical medicine. Cell size is an important indicatorof pathology and in conjunction with haemoglobin, forms the basicclassification of anemias. It is the basis of the differentiation ofwhite blood cells and leukaemias, the assessment of prognosis in scoresof diseases and is an invaluable screening device for the thalassaemias,the most common genetic disease in the world.

[0005] Existing methodology achieves good cell size measurements in themajority of patients, failing only in those whose red cell shape differssignificantly from normal and in patients with abnormal serumosmolality. As cell shape is normally distributed, 4.56% of thepopulation have red cells that deviate from normal cell shape by morethan two standard deviations. These abnormal samples are overrepresented in hospitals because the deviations are associated withillness more often than those within normal limits. Consequently,clinical laboratories generate incorrect cell volume values in at least4.56% of reported results, representing about 100,000 incorrect cellvolume measurements in the United States each day. In the majority ofpatients, these results do not cause significant problems butoccasionally the erroneous measurements result in misdiagnosis andpatient's death. However, this error is rarely recognised becauseexisting automated methods cannot detect it.

[0006] A normal human blood sample is isotonic with a solution having anosmolality of about 290 mosm Kg⁻¹, and at this osmolality the averagered blood cell in the average individual will have a biconcave shape. Itis well known that reducing the osmolality of the solution surrounding ared blood cell below a critical level will cause that cell to swell,then rupture, forming a ghost cell which slowly releases its contents,almost entirely haemoglobin, into the surrounding medium. This process,called haemolysis, can be induced using water (osmotically) or bydetergents, venoms or other chemicals, thermal, mechanical or electricalagents. Tests to determine cell volume are typically carried out atisotonic osmolality.

[0007] Automated measures of cells depend upon both size and shape ofthe cells being tested. Since existing instruments cannot determine cellshape, instruments such as the particle counter sold under the TradeMark Coulter Counter by Coulter Electronics Inc., compensate in thecalculation of isotonic cell volume with a term that is a fixed averageestimate that is in error whenever the cell shape in a sample deviatesfrom the normal population. It is these abnormal samples that indicatepathology where accuracy is most needed that the method most oftenfails.

[0008] In order to estimate the size and the count of the number andproperties of red blood cells in a sample, there are several commercialparticle counters available which may be used. These particle countersact either by measuring the electrical or optical properties of a streamof cells that pass along a narrow tube. The property measured is usuallythe current flowing through the suspension in the tube or the electricalfield within the tube. The signal generated depends upon several factorsincluding cell size, cell shape and the properties of the cell membranethe difference in the electrical property of the cell and the suspendingmedium.

DISCLOSURE OF INVENTION

[0009] According to a first aspect of the present invention, there isprovided a new method in which a sample of cells suspended in a liquidmedium, wherein the cells have at least one measurable property distinctfrom that of the liquid medium, is subjected to analysis by a methodincluding the steps of:

[0010] (a) passing a first aliquot of the sample cell suspension througha sensor,

[0011] (b) measuring said at least one property of the cell suspension,

[0012] (c) recording the measurement of said property for the firstaliquot of cells,

[0013] (d) subjecting the first or at least one other aliquot of thesample cell suspension to an alteration in at least one parameter of thecell environment which has the potential to alter the shape of the cellsto a known or identifiable extent to create an altered cell suspension,

[0014] (e) passing said altered cell suspension through a sensor,

[0015] (f) measuring said at least one property of the altered cellsuspension,

[0016] (g) recording the measurement of said at least one property forsaid altered suspension,

[0017] (h) comparing the data from steps (c) and (g) and determining ashape compensation factor to be applied to the measurement of said atleast one property of the first aliquot of cells in step (c) in thecalculation of a cell parameter to take account of a variation in shapebetween the first aliquot of cells in step (c) and said altered cellsuspension in step (g).

[0018] In the present invention, a cell parameter, for example cellvolume, is determined by subjecting one or more aliquots of a samplecell suspension to one or more alterations of at least one parameter ofthe cell environment to identify a point at which the cells achieve aparticular shape to obtain a sample specific shape compensation factor.

[0019] All existing automated methods include a fixed shape correctionin the treatment of sensor readings taken from a single cell suspensionin which the cell environment is not altered during the course of thetest, which compensates for the deviation of the cells from sphericalshape particles commonly used to calibrate the instruments. However, ina calculation of cell volume, as the cell shape is unknown, a fixedcorrection of approximately 1.5 is entered into the calculation on theassumption that a sample cell has the shape of a biconcave disc. Thiscorrection is correct for the average cell in the average person atisotonic osmolality, but it is incorrect for many categories of illnesswhere the assumed fixed correction may induce an error of up to 60% inthe estimate of cell volume. In the method of the present invention, anestimate is made of the in vivo cell shape so that a true estimate ofcell volume or other cell parameter at all shapes is obtained. In thepreferred embodiment of the present invention, a shape correctionfunction is determined which is used to generate a shape correctionfactor which is a measure of the shape of the cell specific for thatcell sample. The value of the shape correction factor generated by thisfunction then replaces the conventional fixed shape correction of 1.5 toobtain a true measure of cell volume and other cell parameters.

[0020] According to a second aspect of the present invention, anapparatus for testing a sample cell suspension in a liquid medium inaccordance with the method of the first aspect of the present inventioncomprises data processing means programmed to compare data from saidsteps (c) and (g) to determine a shape compensation factor to be appliedto the measurement of said at least one property of the first aliquot ofcells in the calculation of a cell parameter to take account of avariation in shape between the first aliquot of cells and said alteredcell suspension.

[0021] Preferably, the data processing means comprises the internalmicroprocessor of a personal computer.

[0022] Preferably, the property of the cells which differs from theliquid medium is one which is directly related to the volume of thecell. Such a property is electrical resistance or impedance, and this ismeasured as in the normal Coulter Counter by determining the flow ofelectrical current through the cell suspension as it passes through asensing zone of the sensor. The sensing zone is usually a channel oraperture through which the cell suspension is caused to flow. Any typeof sensor may be used provided that the sensor produces a signal whichis proportional to the cell size. Such sensor types may depend uponvoltage, current, RF, NMR, optical, acoustic or magnetic properties.Most preferably, the sensor is substantially as described in ourco-pending International application also filed this day (Agents'reference: 62/2681/03).

[0023] Although the method is usually carried out on blood cells, forinstance white or, usually, red blood cells, it may also be used toinvestigate other cell suspensions, which may be plant or animal cellsor micro-organism cells, for instance, bacterial cells.

[0024] The environmental parameter which is changed in the method may beany change which will result in a measurable parameter of the cellsbeing altered. The method is of most value where the change inenvironmental parameter changes the size, shape, or other anatomicalproperty of the cell. The method is of particular value in detecting achange in the volume of cells as a result of a change of osmolality ofthe surrounding medium. Preferably therefore, the environmentalparameter change is an alteration, usually a reduction, in osmolality.Typically the environment of the first aliquot is isotonic, and thus theenvironment of the altered suspension in step (g) is rendered hypotonic,for instance by diluting a portion of isotonic sample suspension with ahypotonic diluent.

[0025] The method of the present invention, as well as being applicableto cells, as described above, may also be applicable to other naturaland synthetic vesicles which comprise a membrane surrounding an interiorspace, the shape or size or deformability of which may be altered byaltering an environmental parameter. Such vesicles may be useful asmembrane models, for instance, or as drug delivery devices or as devicesfor storing and/or stabilising other active ingredients or to containhaemoglobin in blood substitutes.

[0026] In the method, the time between the initiation of the alterationof the environment to the passage of the cells through the sensing zonemay vary but preferably is less than 1 minute, more preferably less than10 seconds. The time is generally controlled in the method andpreferably it is kept constant. If it changes, then time may be afurther factor which is taken into account in the calculation step ofstep (h).

[0027] Although it is possible for the method of the invention tocomprise merely of the treatment of two aliquots of the sample cellsuspension, more usually the method includes the steps of subjectinganother aliquot of sample cell suspension to a second alteration in atleast one parameter of the cell environment passing said altered aliquotthrough the sensor, recording the change in said property of the cellsuspension under the altered environment as each of a number of cells ofthe aliquot passes through the sensor, recording all the concomitantproperties of the environment together with the said change on acell-by-cell basis, and comparing the data from previous step (c) andthe preceding step as a function of the extent of said second alterationof environmental parameter. Usually there are many further aliquotstreated in a similar way. The greater the number of aliquots tested, thegreater the potential accuracy, precision and resolution of the resultswhich are obtained. It is also possible to subject a only single aliquotof sample suspension to a series of such alterations in at least oneparameter of the cell environment.

[0028] In its simplest form, the test is dependent upon two sensormeasurements, one of which is at a maximum, or near to it. However, theenvironment required to induce a cell to reach a maximum size can beentirely unknown. Furthermore, the environmental changes can besequential, non-sequential, non-sequential, random, continuous ordiscontinuous, provided that the maximum achievable cell size isrecorded. One convenient way of ensuring this is to test the cell in acontinuously changing environment so that all possible cell sizes arerecorded, including the maximum.

[0029] The second alteration in the cell environment is usually of thesame type as the first alteration. It may even be of the same extent asthe first alteration, but the time between initiation of the alterationand passage of the cells through the sensing zone may be different,thereby monitoring the rate of change in the cells properties whensubjected to a particular change in environmental parameter. Thistechnique may also be used to monitor cells which have been in storagefor several years.

[0030] In another embodiment the second alteration in environmentalparameter is of the same type as the first alteration, but has adifferent extent. In such a case, it is preferred for the time betweeninitiation of the alteration and passage of the cells through thesensing zone to be the same for each aliquot of the cell suspension.Preferably, in this embodiment of the method second and subsequentaliquots of cell suspension are subjected to successively increasingextents of alteration of the environmental parameter such that thechange of said property produces a maximum and then decreases as theextent of alteration of environmental parameter is increased. In thepreferred embodiment in which the property of the cell suspension whichis monitored is directly related to the volume of the cells, and wherethe alteration of environmental parameter for the second and subsequentaliquots results in a volume increase of the cells, preferably, theenvironmental change is varied until the cell volume passes a maximum.

[0031] Since the preferred application of the method of the presentinvention is to analyse red blood cells, the following discussion isbased mainly on the study of such cells. It will be realised, however,that the method is, as mentioned above, applicable to other cell typesand to determine other information concerning an organism from a studyof such cell types.

[0032] In current practice, cell shape, particularly red blood cellshape, is not estimated by any automated method. The present inventionenables the user to determine cell shape and derive other data, such ascell volume, surface area, surface area to volume ratio, sphericityindex, cell thickness, and surface area per milliliter. Aside fromresearch and experimental laboratories, none of these measurements arecurrently available in any clinical laboratory and hitherto, none couldbe completed within 60 seconds. In particular, the preferred methodwhere the sample cell suspension is subjected to a concentrationgradient, enables the automatic detection or a user to detect accuratelywhen the cells adopt a substantially spherical shape immediately beforelysis.

[0033] The commercially available Coulter Counter particle counterinstrument produces a signal in proportion to the volume of particleswhich pass through a sensing zone, typically a voltage pulse for eachparticle. The size of the signal is calibrated against spherical latexparticles of known volume to produce a conversion factor to convert ameasured signal, typically voltage, into a particle volume, typicallyfemtoliters. When using particle counters of this type to measure thesize of particles that are not spheres, as is typical in biologicalsamples such as platelets, fibroblasts or red blood cells which have theshape of a disc, a fixed shape correction factor is used in addition tothe conversion factor. This fixed shape correction, based on theoreticaland empirical data, is designed to produce a correct volume estimatewhen measuring particles that are not spherical as the size of thevoltage pulses are not solely related to cell volume. For instance,normal red blood cells produce sensor pulses which are too small by afactor of around 1.5 when measured on these instruments and therefore afixed correction of 1.5 is entered into the calculation of cell volumeto produce the correct valve.

[0034] In the preferred method of the present invention, this fixedshape correction factor is replaced with a sample specific shapecorrection factor f(K_(shape)), generated from a shape correctionfunction. The shape correction function is continuous for all cellshapes and ranges in value from 1.0 for spherical cells to infinity fora perfectly flat cell. The shape correction function increases theaccuracy with which cell parameters which depend on anatomicalmeasurement, such as cell volume, can be determined.

[0035] In the preferred method where the environmental parameter changeis a reduction in osmolality, the following general function describes ashape correction factor based on any two sensor readings ie. measuredvoltages:

f(K _(shape))=f(SR1, SR2)

[0036] where SR1 is a sensor reading (measured voltage) at a knownshape, typically spherical, and SR2 is a sensor reading at an osmolalityof interest, typically isotonic. In the preferred method, the sensorreading is one of voltage amplitude.

[0037] Analysis has shown that this is a linear function and that:${f\left( K_{shape} \right)} = {1 + {\left\lbrack \frac{\left( {{S\quad R\quad 1} - {S\quad R\quad 2}} \right)}{\left( {S\quad R\quad 1} \right)} \right\rbrack \times K_{a}}}$

[0038] where K_(a) is an apparatus dependent constant.

[0039] Preferably, the shape correction factor a blood cell isdetermined by comparing the measured voltage (SR1) with the measured(SR2) voltage of cells of the same blood sample at some known oridentifiable shape, most conveniently cells which have adopted aspherical shape.

[0040] According to a third aspect of the present invention there isprovided a new method in which a sample of cells suspended in a liquidmedium, wherein the cells have at least one measurable property distinctfrom that of the liquid medium, is subjected to analysis by a methodincluding the steps:

[0041] (a) passing a first aliquot of the sample cell suspension througha sensor,

[0042] (b) measuring said at least one property of the cell suspensionas each of a number of cells of the first aliquot passes through thesensor,

[0043] (c) recording the measurement of said property for the firstaliquot of cells on a cell-by-cell basis,

[0044] (d) subjecting the first or at least one other aliquot of thesample cell suspension to an alteration in at least one parameter of thecell environment which has the potential to alter the said at least oneproperty of the cells to create an altered cell suspension,

[0045] (e) passing said altered cell suspension through a sensor,

[0046] (f) measuring said at least one property of the altered cellsuspension as each of a number of cells of the altered cell suspensionpasses through the sensor,

[0047] (g) recording the measurement of said at least one property forthe altered cell suspension on a cell-by-cell basis,

[0048] (h) comparing the data from steps (c) and (g) as a function ofthe extent of said alteration of said parameter of the cell environmentand frequency distribution of said at least one property.

[0049] By carrying out the method of the invention, and in particular byrecording the property change data for the cells on a cell-by-cellbasis, the data can be subsequently treated so as to identifysub-populations of cells within the sample which respond differently toone another under the imposition of the environmental parameteralteration.

[0050] The present invention provides a method for testing blood sampleswhich enables data to be obtained on a cell-by-cell basis. By using thedata on a cell-by-cell basis, it enables new parameters to be measuredand to obtain information on the distribution of cells of differentsizes among a population and reveal sub-populations of cells based ontheir anatomical and physiological properties.

[0051] A measure of reproducibility is the standard deviation of theobservations made. An aspect of the present invention is to provideimprovements in which the standard deviation of the results obtained isreduced to ensure clinical utility.

[0052] According to a fourth aspect of the present invention, anapparatus for testing a sample cell suspension in a liquid medium inaccordance with the method of the third aspect of the present inventioncomprises data processing means programmed to compare data from saidsteps (c) and (g) as a function of the extent of said alteration of saidparameter of the cell environment and frequency distribution of said atleast one property.

[0053] Other environmental parameter changes which may be investigatedinclude changes in pH, changes in temperature, pressure, ionophores,changes by contact with lytic agents, for instance toxins, cell membranepore blocking agents or any combinations of these parameters. Forinstance, it may be useful to determine the effectiveness of lyticagents and/or pore blockers to change the amount or rate of cell volumechange on a change in environmental parameters such as osmolality, pH ortemperature. Furthermore the effects of two or more agents which affecttransport of components in or out of cells on one another may bedetermined by this technique. It is also possible to subject the cellsuspension to a change in shear stress during the passage of the cellsuspension through the sensing zone by changing the flow rate throughthe sensor, without changing any of the other environmental parametersor in conjunction with a change in other environmental parameters. Achange in the shear stress may affect the shape of the cell and thus theelectrical, optical or other property which is measured by the sensor.Monitoring such a change in the deformation of cells may be of value. Inparticular, it may be of value to monitor the change in deformabilityupon changes imposed by disease or, artificially by changing otherenvironmental parameters, such as chemical components of the suspendingmedium, pH, temperature or osmolality.

[0054] Preferably, the data processing means comprises the internalmicroprocessor of a personal computer.

[0055] When full data are available on the distribution of cell size ina particular population of cells subjected to haemolytic shock in a widerange of hypotonic solutions, at osmolalities just below the criticalosmolality causing lysis, a gap in the populations is visible. On a 3-Dplot or an alternative way of representing the data such as a contourmap, the ghost cells are clearly visible and the unruptured cells areclearly identifiable, but between them there is a region defined by, forexample, osmolality and cell size where the cells are widelydistributed. The existence of this phenomenon, which we have termed“ghost gap”, has not previously been recognised, and it has beendiscovered that the nature of this phenomenon varies with species andbetween healthy and diseased individuals of particular species. It is ameasure of the degree of anisocytosis (size heterogeneity) and can beused in the measurement of the degree of poikilocytosis (shapeheterogeneity) of the cell population, which is often used as the basisfor classifying all anaemia.

[0056] The measurements of the cell parameter changes may be stored andretrieved as voltage pulses and they may be displayed as individual dotson a display of voltage against the osmolality of the solution causingthe parameter change. When observations are made using a suspension at asingle tonicity, the resulting plot shows the frequency distribution ofvoltage by the intensity of the dots representing cells of the samevolume.

[0057] The number of blood cells within each aliquot which are countedis typically at least 1000 and the cell-by-cell data is then used toproduce an exact frequency distribution of size. Suitably this densitycan be made more visible by using different colours to give a threedimensional effect, similar to that seen in radar rainfall pictures usedin weather forecasting. Alternatively, for a single solution of anytonicity, the measured parameter change could be displayed against thenumber of individual cells showing the same change. In this way adistribution of cell volume or voltage in a particular tonicity of givenosmolality can be obtained.

[0058] The method of the invention may be further improved by, insteadof subjecting portions of a sample each to one of a series of hypotonicsolutions of different osmolalities to form the individual aliquots, thesample is fed continuously into a solution, the osmolality of which ischanged continuously to produce a continuous gradient of aliquots forpassage through the sensing zone. Preferably, identical portions of thesample under test are subjected to solutions of each osmolalitythroughout the range under test after the same time from imposition ofthe environmental parameter change to the time of passage through thesensing zone. This technique ensures that the cells are subjected to theexact concentration which cause critical changes in that particularsample. Further, an effect of feeding the sample under test into acontinuously changing osmolality gradient, is to obtain measurementswhich are equivalent to treating one particular cell sample with thatcontinuously changing gradient. This technique is the subject of ourco-pending International patent application also filed this day (Agents'reference: 62/2684/03).

[0059] Further, in the present invention, it is possible to examine aparticular blood sample at various intervals of time and compare thesets of results to reveal dynamic changes in cell function.

[0060] These dynamic changes have revealed that cells slowly decreasetheir ability to function over time, but they also change in unexpectedways. The size and shape of the cells in a blood sample change in acomplex, non-linear but repeatable way, repeating some of thecharacteristic patterns of change over the course of days and onsuccessive testing. The patterns, emerging over time, show similarityamong like samples and often show a characteristic wave motion. Thepattern of change may vary between individuals reflecting the health ofthe individual, or the pattern may vary within a sample. Thus a samplethat is homogeneous when first tested may split into two or severalsub-populations which change with time and their existence can bedetected by subjecting the sample to a wide range of differenttonicities and recording the cell size in the way described.

[0061] If the entire series of steps are repeated at timed intervals onfurther aliquots of the original sample and the resulting propertychange is plotted against osmolality, time and frequency distribution, afour-dimensional display, is obtained which may be likened to a changingweather map. The rate of change of the property in relation to the timetaken to perform each test must be such that any changes which occurduring the test must not substantially affect the results.

[0062] It is this moving three-dimensional display (motion in time beingthe fourth dimension), which provides a pattern characteristic of aparticular blood sample. The pattern includes the density of particularcell voltages and thereby sizes, the shape of the area representingghost cells and particularly the shape and location of a gap between thewhole cells and the ghost cells. This pattern and its variation withspecies and the health of individuals within a species provides acharacteristic which may be used in clinical diagnosis and otherdisciplines. Cell shape is one property that is the basis of thisdancing or changing display. For the first few hours the cell becomesincreasingly spherical in the original sample, it then becomes flatterfor several hours, then more spherical again reaches a limit and thenbecomes thinner and finally may swell again. This set of curious changesin shape also occurs within a blood clot and is the basis of thehitherto puzzling changes in opacity of the brain after a strokeobserved by NMR, (1) Gomori J. M., Grossman R. I. et al, Radiology 157,1985, p. 87 to 93; (2) Bydder G. M., Pennock J. M., Porteous R. et al,1988, Neuro-radiology 30, p. 367 to 371; (3) Alanen A., Kormano M. 1985,J. Ultrasound Med 4., 421 to 425. By using the method of the presentinvention it has been determined that the rate at which observed changestake place either within a clot or in a whole blood sample areinfluenced by pH, temperature and available energy.

[0063] The 3D pattern enables identification of the precise osmolalityat which particular cells reach their maximum volume, i.e. when theybecome spheres. With appropriate calibration, and using the magnitude ofthe voltage pulse, it is possible to define precisely and accurately theactual volume of such cells. By causing cells to pass through theirmaximum and differentiating, identification of the point of lysis iesphered cells is obtained to the nearest {fraction (1/10)} mosm Kg⁻¹.When the mean cell volume is required, the data is taken from thevoltages and thereby the volume at isotonic osmolality corrected for theproportion of cells that are more leptocytotic or spherocytic thannormal and the degree of that deviation. When individual cell volumesare required they are obtained directly from the observed voltage pulseor from the frequency distribution data with dispersion statistics orits graphical representation.

[0064] In a preferred form of the invention, when the measurements of ablood sample are made against a continuous gradient of osmolality andthe results displayed as a pattern or contour map of the individual cellmeasurements and showing the distribution of cells of different sizes bythe density of the pattern, repeating this measurement at intervals oftime and then examining each in time sequence reveals that a typicalcell sample may contain sub-populations of cells which change with timein a different way from other cells in that sample. Examination of thepatterns obtained at different points in time is preferably done bycomputer sequencing which gives the effect of the dynamic changes in thecells in the form of a moving picture. Using this technique, two or morecell sub-populations may be followed to determine the fate and therelative proportion of each population. This is useful as a means tomonitor, for example the recovery of the bone marrow in an anaemia thatis under treatment, a leukaemia that has received chemotherapy, a bonemarrow transplant or as a means to study the storage lesion in storedblood.

[0065] According to a further feature of the invention there is provideda method for the diagnosis of abnormalities in a blood cell sample whichcomprises using the invention whereby the data retrieved on anindividual cell basis, or a summary there of, from an experimentalsource is compared with data retrieved on an individual cell basis froma standard sample. It will be clear from the description given abovethat the data available from the improved method of the presentinvention provides a pattern and a method of calculating a number ofindividual blood cell parameters which are capable of comparison withstandard samples. This comparison provides the basis for a wide range ofclinical diagnosis. In particular, the pattern or contour map obtainedby conducting measurements against a continuous gradient of osmolalityprovides a pattern capable of giving a great deal of diagnosticinformation and summaries of such data.

[0066] Attention is also drawn to the use of data obtained bymeasurements carried out at different timed intervals. By viewing thecell population at such timed interval and noting the changes in thepattern which take place, much valuable diagnostic information can beobtained by skilled persons. Again a great deal of work needs to be doneto identify and correlate particular changes in this pattern withclinical conditions. However it is clear that the existence of thisvariation in pattern with time will prove to be a significant diagnostictool.

[0067] The method of the invention may be used in medicine, for examplein clinical diagnosis to detect the presence of disease, or to assessremission and prognosis of a diseased state, or in blood banks to assessthe condition of stored blood. The invention will also have value inveterinary persons. Again a great deal of work needs to be done toidentify and correlate particular changes in this pattern with clinicalconditions. However it is clear that the existence of this variation inpattern with time will prove to be a significant diagnostic tool.

[0068] The method of the invention may be used in medicine, for examplein clinical diagnosis to detect the presence of disease, or to assessremission and prognosis of a diseased state, or in blood banks to assessthe condition of stored blood. The invention will also have value inveterinary medicine for diagnosis and in zoology as a guide to taxonomy.

[0069] This invention has the advantage of being able to detect subtlechanges in red cell morphology, and in particular cell shape changes,long before the cell shows gross changes that may be detectable byexisting methods. For example, gross changes in the red cell thicknesswhich cannot be quantified but can usually be detected by lightmicroscopy, can be quantified using the method of the invention. Furthersmall changes in the red cell thickness cannot be detected or quantifiedby existing methods, but can be both detected and quantified using themethod of the invention.

[0070] The invention has been shown to be useful in the clinicaldiagnosis of many conditions with thin cells and in particular:

[0071] Thin Cells or leptocytes

[0072] 1. Hereditary stomatocytosis

[0073] 2. Mediterranean stomatocytosis

[0074] 3. Cryohydrocytosis

[0075] 4. Adenine deaminase hyperactivity

[0076] 5. Obstructive liver disease

[0077] 6. Rh null blood group

[0078] This invention has been shown to be useful in the clinicaldiagnosis of spherocytic cells in a sample (ie. cell which have more ofa spherical shape than is normal) can be an indication of one of thefollowing:

[0079] 1. ABO haemolytic disease

[0080] 2. Hereditary spherocytosis

[0081] 3. Immunohaemolytic anaemias (Coombs positive)

[0082] 4. Transfusion reaction

[0083] 5. Clostridia infections

[0084] 6. Burns

[0085] 7. Venoms from snakes, spiders or bees

[0086] 8. Hypophosphotaemia

[0087] 9. Hypersplenism

[0088] 10. Pregnancy

[0089] This list gives an indication of the clinical value of thepresent invention which is greatly enhanced once the changes can bequantified and the subtler changes are associated with disease. Theability to measure the degree of thickness or thinness of cells allowsthe possibility to study the progress of the disease and assess thevalue of treatment, especially at an early stage.

[0090] Other parameters identifiable by the method of the invention maybe used to identify other species.

BRIEF DESCRIPTION OF DRAWINGS

[0091] The present invention will now be described in detail withreference to the accompanying drawings, in which:

Detailed Description

[0092]FIG. 1 shows schematically an instrument used to sample and testblood cells;

[0093]FIG. 2 shows velocity profiles for the discharge of fluids fromfluid delivery syringes of a gradient generator section of theinstrument of FIG. 1;

[0094]FIG. 3 shows a block diagram illustrating the data processingsteps used in the instrument of FIG. 1;

[0095]FIG. 4 shows an example of a three-dimensional plot of osmolalityagainst measured voltage for cells of a blood sample analyzed inaccordance with the present invention;

[0096]FIG. 5 shows another example of a three-dimensional plot ofosmolality against measured voltage which illustrates the frequencydistribution of blood cells at intervals;

[0097]FIG. 6 shows a series of three-dimensional plots for a sampletested at hourly intervals;

[0098]FIGS. 7 and 8 show results for spherical latex particles as partof an instrument calibration routine;

[0099]FIG. 9 shows superimposed plots of osmolality (x-axis) againstmeasured voltage and true volume, respectively;

[0100]FIGS. 10a to 10 d show the results from the test of a healthyindividual;

[0101]FIG. 11 shows Price-Jones curves of the results shown in FIGS. 10ato 10 d; and,

[0102]FIG. 12 shows a three-dimensional frequency distribution plot andcell parameters for an abnormal individual.

[0103]FIG. 1 shows schematically the arrangement of a blood sampler foruse in the method of the present invention. The blood sampler comprisesa sample preparation section 1, a gradient generator section 2 and asensor section 3.

[0104] A whole blood sample 4 contained in a sample container 5 acts asa sample reservoir for a sample probe 6. The sample probe 6 is connectedalong PTFE fluid line 26 to a diluter pump 7 via multi-positiondistribution valve 8 and multi-position distribution valve 9. Thediluter pump 7 draws saline solution from a reservoir (not shown) viaport #1 of the multi-position distribution valve 9. As will be explainedin detail below, the diluter pump 7 is controlled to discharge a sampleof blood together with a volume of saline into a first well 10 as partof a first dilution step in the sampling process.

[0105] In a second dilution step, the diluter pump 7 draws a dilutesample of blood from the first well 10 via multi-position distributionvalve 11 into PTFE fluid line 12 and discharges this sample togetherwith an additional volume of saline into a second well 13. The secondwell 13 provides the dilute sample source for the gradient generatorsection 2 described in detail below.

[0106] Instead of using whole blood, a pre-diluted sample of blood 14 ina sample container 15 may be used. In this case, a sample probe 16 isconnected along PTFE fluid line 30, multi-position distribution valve11, PTFE fluid line 12 and multi-position distribution value 9 to thediluter pump 7. In a second dilution step, the diluter pump 7 draws avolume of the pre-diluted sample 14 from the sample container 15 viafluid line 30 and multi-position distribution value 11 into fluid line12 and discharges the sample together with an additional volume ofsaline into the second well 13 to provide the dilute sample source forthe gradient generator section 2.

[0107] The gradient generator section 2 comprises a first fluid deliverysyringe 17 which draws water from a supply via multi-positiondistribution valve 18 and discharges water to a mixing chamber 19 alongPTFE fluid line 20. The gradient generator section 2 also comprises asecond fluid delivery syringe 21 which draws the diluted sample of bloodfrom the second well 13 in the sample preparation section 1 viamulti-position distribution valve 22 and discharges this to the mixingchamber 19 along PTFE fluid line 23 where it is mixed with the waterfrom the first fluid delivery syringe 17. As will be explained in detailbelow, the rate of discharge of water from the first fluid deliverysyringe 17 and the rate of discharge of dilute blood sample from thesecond fluid delivery syringe 21 to the mixing chamber is controlled toproduce a predetermined concentration profile of the sample suspensionwhich exits the mixing chamber 19 along PTFE fluid line 24. Fluid line24 is typically up to 3 meters long. A suitable gradient generator isdescribed in detail in the Applicant's co-pending Internationalapplication also filed this day (Agent's reference 62/2684/03).

[0108] As will also be explained in detail below, the sample suspensionexits the mixing chamber 19 along fluid line 24 and enters the sensorsection 3 where it passes a sensing zone 25 which detects individualcells of the sample suspension before the sample is disposed of via anumber of waste outlets.

[0109] In a routine test, the entire system is first flushed and primedwith saline, as appropriate, to clean the instrument, remove pockets ofair and debris, and reduce carry-over.

[0110] The diluter pump 7 comprises a fluid delivery syringe driven by astepper motor (not shown) and is typically arranged initially to draw 5to 10 ml of saline from a saline reservoir (not shown) via port #1 ofmulti-position distribution valve 9 into the syringe body. A suitablefluid delivery syringe and stepper motor arrangement is described indetail in the Applicant's co-pending application also filed this day(Agents reference 80/4936/01). Port #1 of the multi-positiondistribution valve 9 is then closed and port #0 of both multi-positiondistribution valve 9 and multi-position distribution valve 8 are opened.Typically 100 μl of whole blood is then drawn from the sample container5 to take up the dead space in the fluid line 26. Port #0 ofmulti-position distribution valve 8 is then closed and any blood fromthe whole blood sample 4 which has been drawn into a fluid line 27 isdischarged by the diluter pump 7 to waste via port #1 of multi-positiondistribution valve 8.

[0111] In a first dilution step, port #0 of multi-position distributionvalue 8 is opened and the diluter pump 7 draws a known volume of wholeblood, typically 1 to 20 μl, into PTFE fluid line 27. Port #0 is thenclosed, port #2 opened and the diluter pump 7 discharges the bloodsample in fluid line 27 together with a known volume of saline in fluidline 27, typically 0.1 to 2 ml, into the first well 10. Port #2 ofmulti-position distribution value 8 and port #0 of multi-positiondistribution value 9 are then closed.

[0112] Following this, port #0 of multi-position distribution valve 11and port #3 of multi-position distribution valve 9 are opened to allowthe diluter pump 7 to draw the first sample dilution held in the firstwell 10 to take up the dead space in PTFE fluid line 28. Port #0 ofmulti-position distribution valve 11 is then closed and port #1 openedto allow the diluter pump 7 to discharge any of the first sampledilution which has been drawn into fluid line 12 to waste via port #1.

[0113] In a second dilution step, port #0 of multi-position distributionvalve 11 is re-opened and the diluter pump 7 draws a known volume,typically 1 to 20 μl, of the first sample dilution into fluid line 12.Fluid line 12 includes a delay coil 29 which provides a reservoir toprevent the sample contaminating the diluter pump 7. Port #0 ofmulti-position distribution valve 11 is then closed, port #3 opened, andthe diluter pump 7 then discharges the first sample dilution in fluidline 12, together with a known volume of saline, typically 0.1 to 20 ml,into the second well 13. Port #3 of multi-position distribution valve 11is then closed. At this stage, the whole blood sample has been dilutedby a ratio of typically 10000:1. As will be explained below, theinstrument is arranged automatically to control the second dilution stepto vary the dilution of the sample suspension to achieve a predeterminedcell count to within a predetermined tolerance at the start of a testroutine.

[0114] In the gradient generator section 2, the first fluid deliverysyringe 17 is primed with water from a water reservoir. Port #3 ofmulti-position distribution valve 22 is opened and the second fluiddelivery syringe draws a volume of the dilute blood sample from thesecond well 13 into the syringe body. Port #3 of multi-positiondistribution valve 22 is then closed and port #2 of both multi-positiondistribution valve 18 and multi-position distribution valve 22 areopened prior to the controlled discharge of water and dilute bloodsample simultaneously into the mixing chamber 19.

[0115]FIG. 2 shows how the velocity of the fluid discharged from each ofthe first and second fluid delivery syringes is varied with time toachieve a predetermined continuous gradient of osmolality of the samplesuspension exiting the mixing chamber 19 along fluid line 24. The flowrate of the sample suspension is typically in the region of 200 μl s⁻¹which is maintained constant whilst measurements are being made. Thisfeature is described in detail in the Applicant's co-pending application(Agent's reference 62/2684/01). As shown in FIG. 2, a cam profileassociated with a cam which drives fluid delivery syringe 21 acceleratesthe syringe plunger to discharge the sample at a velocity V₁, whilst acam profile associated with a cam which drives fluid delivery syringe 17accelerates the associated syringe plunger to discharge fluid at a lowervelocity V₂. Once a constant flow rate from each delivery syringe hasbeen established at time T_(o), at time T₁ the cam profile associatedwith fluid delivery syringe 21 causes the rate of sample discharge todecelerate linearly over the period T₂-T₁ to a velocity V₂, whilesimultaneously, the cam profile associated with fluid delivery syringe17 causes the rate of fluid discharge to accelerate linearly to velocityV₁. During this period, the combined flow rate of the two syringesremains substantially constant at around 200 μls⁻¹ Finally, the twosyringes are flushed over the period T₃-T₂.

[0116] Once both the first fluid delivery syringe 17 and the secondfluid delivery syringe 21 have discharged their contents, the firstdelivery syringe is refilled with water in preparation for the nexttest. If a blood sample from a different subject is to be used, thesecond fluid delivery syringe 21 is flushed with saline from a salinesupply via port #1 of multi-position distribution valve 22 to clean thecontaminated body of the syringe.

[0117] The sample suspension which exits' the mixing chamber 19 passesalong fluid line 24 to the sensor section 3. A suitable sensor sectionis described in detail in the Applicant's co-pending Internationalapplication also filed this day (Agent's reference 62/2681/03). Thesample suspension passes to a sensing zone 25 comprising an electricalfield generated adjacent an aperture through which the individual cellsof the sample suspension must pass. As individual blood cells of thesample suspension pass through the aperture the response of theelectrical field to the electrical resistance of each individual cell isrecorded as a voltage pulse. The amplitude of each voltage pulsetogether with the total number of voltage pulses for a particularinterrupt period, typically 0.2 seconds, is also recorded and stored forsubsequent analysis including a comparison with the osmolality of thesample suspension at that instant which is measured simultaneously. Theosmolality of the sample suspension may also be determined withoutmeasurement from a knowledge of the predetermined continuous osmoticgradient generated by the gradient generator section 2. As describedbelow, the osmolality (pressure) is not required to determine the cellparameters.

[0118]FIG. 3 shows how data is collected and processed. Inside eachinstrument is a main microprocessor which is responsible for supervisingand controlling the instrument, with dedicated hardware or low-costembedded controllers responsible for specific jobs within theinstrument, such as operating diluters, valves, and stepper motors ordigitizing and transferring a pulse to buffer memory. The software whichruns the instrument is written in C and assembly code and is slightlyless than 32 K long.

[0119] When a sample is being tested, the amplitude and length of eachvoltage pulse produced by the sensor is digitized to 12-bit precisionand stored in one of two 16K buffers, along with the sum of theamplitudes, the sum of the lengths, and the number of pulses tested.Whilst the instrument is collecting data for the sensors, one buffer isfilled with the digitized values while the main microprocessor emptiesand processes the full buffer. This processing consists of filtering outunwanted pulses, analysing the data to alter the control of theinstrument and finally compressing the data before it is sent to thepersonal computer for complex analysis.

[0120] Optional processing performed by the instrument includes digitalsignal processing of each sensor pulse so as to improve filtering,improve the accuracy of the peak detection and to provide moreinformation about the shape and size of the pulses. Such digital signalprocessing produces about 25 16-bit values per cell, generating about 25megabytes of data per test.

[0121] Data processing in the personal computer consists of a custom 400K program written in C and Pascal. The PC displays and analyses the datain real time, controls the user interface (windows, menus, etc.) andstores and prints each sample.

[0122] The software also maintains a database of every sample testedenabling rapid comparison of any sample which has been previouslytested. Additionally, the software monitors the instrument's operationto detect malfunctions and errors, such as low fluid levels, systemcrashes or the user forgetting to turn the instrument on.

[0123] The voltage pulse generated by each cell of the sample suspensionas it passes through the aperture of sensing zone 25 is displayed ingraphical form on a VDU of a PC as a plot of osmolality against measuredvoltage. The sample suspension passes through the sensor section at arate of 200 μls⁻¹. The second dilution step is controlled to achieve aninitial cell count of around 5000 cells per second, measured at thestart of any test, so that in an interrupt period of 0.20 seconds,around 1000 cells are detected and measured. This is achieved by varyingautomatically the volume of saline discharged by the diluter pump 7 fromthe fluid line 12 in the second dilution step. Over a test period of 40seconds, a total of 200 interrupt periods occur and this can bedisplayed as a continuous curve in a three-dimensional form toillustrate the frequency distribution of measured voltage at anyparticular osmolality, an example of which is shown in FIGS. 4 and 5.

[0124] The measured cell voltage, stored and retrieved on an individualcell basis is shown displayed on a plot of voltage against theosmolality of the solution causing that voltage change. Using individualdots to display the measured parameter change for each individual cellresults in a display whereby the distribution of cells by voltage, andthereby by volume, in the population is shown for the whole range ofsolutions covered by the osmolality gradient. The total effect is athree-dimensional display shown as a measured property change in termsof the amplitude of the measured voltage pulses against alteredparameter, in this case the osmolality of the solution, to which thecells have been subjected and the distribution or density of the cellsof particular sizes within the population subjected to the particularosmolality. The effect is to produce a display analogous to a contourmap, which can be intensified by using colour to indicate the areas ofgreatest intensity.

[0125] When full data is available on the distribution of cell size in aparticular population of cells subjected to haemolytic shock in a widerange of hypotonic solutions, at osmolalities just below a criticalosmolality causing lysis a gap in the populations is visible. As shownin FIG. 4, ghost cells are fully visible or identifiable in thethree-dimensional plot and the unruptured cells are clearlyidentifiable, but between them is a region defined by osmolality andcell volume where relatively few individuals appear. The existence ofthis phenomenon, which we have termed the “ghost gap”, has notpreviously been recognised.

[0126] If the entire series of steps are repeated at timed intervals onfurther aliquots of the original sample and the resulting measuredvoltage is plotted against osmolality, time and frequency distribution,a four-dimensional display, is obtained which may be likened to a changein weather map. This moving three-dimensional display, its motion intime being the fourth dimension, provides an additional patterncharacteristic of a particular blood sample. This is shown in the seriesof images in FIG. 6. The images shown in FIG. 6 are the results of testscarried out at hourly intervals at a temperature of 37° C. As themeasurements are so exact, the repeat values are superimposable usingcomputer sequencing techniques.

[0127] As shown, cells slowly lose their ability to function over time,but they also change in unexpected ways. The size and shape of the cellsin a blood sample change in a complex, non-linear but repeatable way,repeating some of the characteristic patterns over the course of daysand on successive testing. The patterns, emerging over time, showsimilarity among like samples and often show a characteristic wavemotion. The pattern of change may vary between individuals reflectingthe health of the individual, or the pattern may vary within a sample.Thus a sample that is homogeneous when first tested may split into twoor several sub-populations which change with time and their existencecan be detected by subjecting the sample to a wide range of differenttonicities and recording the voltage pulse in the way described. Asshown in FIG. 6, after the first few hours the cell becomes increasinglyspherical in the original sample, it then becomes flatter for severalhours, then more spherical again, reaches a limit, and then becomesthinner and finally may swell again. It has been determined that therate at which observed changes take place are influenced by pH,temperature, available energy and other factors.

[0128] The three-dimensional pattern provides data which enablesidentification of the precise osmolality at which particular cells reachtheir maximum volume, when they become spheres. With appropriatecalibration, which is described in detail below, and using the magnitudeof the voltage pulse, it is possible to define precisely and accuratelythe actual volume of such cells and thereafter derive a number of othercell parameters of clinical interest.

[0129] The amplitude of the voltage pulses produced by the sensor 25 asindividual cells pass through the electrical field are proportional tothe volume of each cell. However, before a conversion can be performedto provide a measure of cell volume, the instrument requirescalibration. This is performed using spherical latex particles of knownvolume and by comparison with cell volumes determined using conventionaltechniques.

[0130] Experimental results have shown that the mapping of measuredvoltage to spherical volume of commercially available latex particles isa linear function. Accordingly, only a single size of spherical latexparticles needs to be used to determine the correct conversion factor.In a first calibration step, a sample containing latex particlesmanufactured by Bangs Laboratories Inc. having a diameter of 5.06 μmi.e. a volume of 67.834 m³, was sampled by the instrument. Thethree-dimensional plot for the latex particles is shown in FIG. 7 with aplot of osmolality against mean voltage shown in FIG. 8. In thisparticular test, the instrument produced a mean voltage of 691.97 mV.The spherical volume is given by the equation:

Spherical volume=measured voltage×K_(volts)

[0131] where K_(volts) is the voltage conversion factor.

[0132] Re-arranging this equation gives:$K_{volts} = \frac{{spherical}\quad {volume}}{{measured}\quad {voltage}}$

[0133] which in this case gives,$K_{volts} = {\frac{67.834}{691.97} = 0.0980}$

[0134] This value of K_(volts) is only valid for the particularinstrument tested and is stored in a memory within the instrument.

[0135] In a second calibration step, a shape correction factor isdetermined to take account of the fact that the average blood cell inthe average individual has a bi-concave shape. Applying the abovevoltage conversion factor K_(volts) assumes that, like the latexparticles, blood cells are spherical and would therefore give anincorrect cell volume for cell shapes other than spherical. In thepresent invention, a variable shape correction function is determined sothat the mean volume of the blood cells at any osmolality up to thecritical osmolality causing lysis can be calculated extremelyaccurately.

[0136] To illustrate this, a sample was tested at a number of accuratelyknown osmolalities and the volume of the blood cells measured using astandard reference method, packed cell volume. A portion of the samesample was also tested by the method of the present invention using theinstrument of FIG. 1 to measure the voltage pulses from individual cellsat the corresponding osmolalities. The results of these procedures areshown in Table 1 and plotted as two superimposed graphs of osmolality(x-axis) against measured voltage and true volume, respectively, in FIG.9.

[0137] At an isotonic osmolality of 290 mosm, the true volume, asdetermined by the packed cell volume technique, was 92.0 fl, whilst themeasured mean voltage was 670 mV.

[0138] The true isotonic volume of the cells is given by equation:

Volume_(iso)=Voltage_(iso)×K_(volts)×K_(shape)

[0139] where Voltage_(iso) is the measured voltage and K_(shape) is ashape correction factor.

[0140] Re-arranging:$K_{shape} = \frac{{Volume}_{iso}}{{Voltage}_{iso} \times K_{volts}}$

[0141] which in this example gives,$K_{shape} = {\frac{92.0}{670 \times 0.0980} = 1.4}$

[0142] Table 1 shows the shape correction factor K_(shape) for each ofthe other aliquots and demonstrates that the factor to be applied toeach sample is different with the maximum shape correction being appliedat isotonic osmolalities where the blood cells are bi-concave ratherthan spherical. To automate the calculation of K_(shape) at anyosmolality of interest a shape correction function is required. Thefollowing general function describes a shape correction factor based onany two sensor readings i.e. measured voltages:

f(K_(shape))=f(SR1, SR2)

[0143] where SR1 is a sensor reading (measured voltage) at a knownshape, typically spherical, and SR2 is a sensor reading (measuredvoltage) at an osmolality of interest, typically isotonic.

[0144] Analysis has shown that this is a linear function and that:${f\left( K_{shape} \right)} = {1 + {\left\lbrack \frac{\left( {{S\quad R\quad 1} - {S\quad R\quad 2}} \right)}{\left( {S\quad R\quad 1} \right)} \right\rbrack \times K_{a}}}$

[0145] where K_(a) is an apparatus dependent constant, which isdetermined as follows:

[0146] K_(shape) at an osmolality of 290 mosm is known (see above),applying the values SR1=1432 mV, SR2=670 mV and K_(shape)=1.4 to theabove equation gives:$1.4 = {1 + {\left\lbrack \frac{\left( {1432 - 670} \right)}{1432} \right\rbrack \times K_{a}}}$

[0147] re-arranging:

K_(a)=0.7518

[0148] This value of K_(a) is constant for this instrument.

[0149] The true isotonic volume of a blood sample is determined bycomparing the measured voltage at an isotonic volume of interest withthe measured voltage of cells of the same blood sample at some known oridentifiable shape, most conveniently cells which have adopted aspherical shape, whereby:

Volume_(iso)=Voltage_(iso) ×K _(volts) ×f(K _(shape))

[0150]${Volume}_{iso} = {{{Voltage}_{iso} \times K_{Volts} \times {f\left( K_{shape} \right)}} = {S\quad R\quad 2 \times 0.0980 \times \left\lbrack {1 + {\left\lbrack \frac{\left( {{S\quad R\quad 1} - {S\quad R\quad 2}} \right)}{S\quad R\quad 1} \right\rbrack \times 0.7518}} \right\rbrack}}$

[0151] In the present invention, the point at which the blood cellsbecome spherical when subjected to a predetermined continuous osmoticgradient can be determined very accurately. FIGS. 10a-10 d show theresults for a normal blood sample from a healthy individual. FIG. 10ashows a three-dimensional plot of measured voltage against osmolality,FIG. 10b shows a graph of osmolality against percentage change inmeasured voltage for a series of tests of a sample, FIG. 10c shows theresults in a tabulated form, and FIG. 10d shows superimposed graphs ofmean voltage and cell count for the test, respectively, againstosmolality. As shown, the cell count, which is initially 5000 cells persecond at the beginning of a test, reduces throughout the test due tothe dilution of the sample in the gradient generator section 2. The meanvoltage rises to a maximum at a critical osmolality where the bloodcells achieve a spherical shape and then reduces. Using standardstatistical techniques, the maxima of the curve in FIG. 10b, andtherefore the mean voltage at the maxima, can be determined. The meanvoltage at this point gives the value SR1 for the above equation. It isthen possible to select any osmolality of interest, and the associatedmeasured voltage SR2, and calculate the true volume of the cell at thatosmolality. Typically, the isotonic osmolality is chosen, correspondingto approximately 290 mosm.

[0152] For the above test, at 290 mosm, SR1=1432 mV and SR2=670 mV.Accordingly:${f\left( K_{{shape}\quad 290} \right)} = {1 + {\left\lbrack \frac{1432 - 670}{1432} \right\rbrack \times 0.7518}}$

[0153] K_(shape 290)=1.40

[0154] and therefore: $\begin{matrix}{{Volume}_{iso} = {S\quad R\quad 2 \times K_{Volts} \times K_{shape}}} \\{= {670 \times 0.0980 \times 1.40}} \\{= {91.92\quad {fl}}}\end{matrix},\text{and:}$ $\begin{matrix}{{Volume}_{sph} = {S\quad R\quad 1 \times K_{Volts} \times K_{shape}}} \\{= {1432 \times 0.098 \times 1.0}} \\{= {140.34\quad {fl}}}\end{matrix}.$

[0155] Knowledge of the mean volume of the sphered cells allowscalculation of spherical radius as:${Volume}_{sph} = \frac{4\pi \quad r^{3}}{3}$

[0156] from which the spherical radius$r = \left\lbrack \frac{3 \times {Volume}_{sph}}{4\pi} \right\rbrack^{\frac{1}{3}}$${r = {\left\lbrack \frac{3 \times 140.34}{4\pi} \right\rbrack^{\frac{1}{3}}\quad = {3.22\quad {µm}}}}\quad$

[0157] Having determined volume_(iso), volume_(sph) and the sphericalcell radius, it is possible to calculate a number of other parameters.In particular:

[0158] 1. Surface Area (SA)

[0159] Since the surface area SA is virtually unchanged at allosmolalities, the cell membrane being virtually inelastic, and inparticular between spherical and isotonic, the surface area SA may becalculated by substituting r into the expression: $\begin{matrix}{{SA} = {4\pi \quad r^{2}}} \\{= {4\pi \quad {x(3.22)}^{2}}} \\{= {130.29\quad {µm}^{2}}}\end{matrix}$

[0160] 2. Surface Area to Volume Ratio (SAVR)

[0161] Given that the walls of a red cell can be deformed withoutaltering their area, once the surface area SA is known for a cell or setof cells of any particular shape, the surface area is known for anyother shape, thus the surface area to volume ratio SAVR can becalculated for any volume. SAVR is given by the expression:$\begin{matrix}{{SAVR} = \frac{4\pi \quad r^{2}}{{Volume}_{iso}}} \\{= \frac{SA}{{Volume}_{iso}}} \\{= \frac{130.29}{91.99}} \\{= 1.42}\end{matrix}$

[0162] 3. Sphericity Index (SI)

[0163] The present invention can easily measure the SAVR, a widelyquoted but hitherto, rarely measured indication of cell shape. For aspherical cell, it has the value of 3/r, but since cells of the sameshape but of different sizes may have different SAVR values, it isdesirable to use the sphericity index SI which is a dimensionless unitindependent of cell size, given by the expression: $\begin{matrix}{{SI} = {{SAVR} \times \frac{r}{3}}} \\{= {1.42 \times \frac{3.22}{3}}} \\{= 1.52}\end{matrix}$

[0164] 4. Cell Diameter (D)

[0165] When the normal cell is in the form of a bi-concave disc atisotonic osmolality, it is known that the ratio of the radius of asphere to that of the bi-concave disc is 0.8155. On this basis,therefore, the diameter D of a cell in the form of a bi-concave disc isgiven by: $\begin{matrix}{D = \frac{2r}{0.8155}} \\{= \frac{2 \times 3.22}{0.8155}} \\{= {8.19\quad {µm}}}\end{matrix}$

[0166] The same parameter can be determined for all other osmolalities.The frequency distribution of the cell diameters is given both asdispersion statistics as well as a frequency distribution plot. Thepresent invention provides an automated version of the known manualprocedure of plotting a frequency distribution of isotonic celldiameters known as a Price-Jones curve. The present invention is capableof producing a Price-Jones curve of cell diameters for any shape of celland, in particular, isotonic, spherical and ghost cells (at anyosmolality) and is typically based on 250,000 cells. This is shown inFIG. 10.

[0167] 5. Cell Thickness (CT)

[0168] When the cell is in the form of a bi-concave disc, an approximatemeasure of the cell thickness can be derived from the cross-sectionalarea and the volume. The area is of course derivable from the radius ofthe cell in spherical form. The cell thickness can therefore becalculated as follows: $\begin{matrix}{{CT} = \frac{{Volume}_{iso}}{\pi \quad r^{2}}} \\{= \frac{91.92}{\pi \times 3.22^{2}}} \\{= {2.82\quad {µm}}}\end{matrix}$

[0169] 6. Surface Area per milliliter (SA ml)

[0170] The product of the surface area (SA) and the cell count (RBC) isthe surface area per milliliter (SA ml) available for physiologicalexchange. Typically, a healthy adult male has a value of 1,000,000mm²/ml, or 1 m²/ml. The total surface area of the proximal renal tubesthat are responsible for acid-base regulation of the body fluids is 5m². The total surface area of the red blood cells that also play animportant part in the regulation of the acid-base balance is 4572 m²,almost 3 orders of magnitude larger. RBC is calculated internally from aknowledge, of the flow rate of the diluted blood sample, a cell countfor each sample and the dilution of the original whole blood sample.Typically, RBC is approximately 4.29×10⁹ red cells per ml.$\begin{matrix}{{SAml} = {{SA} \times {RBC}\quad \left( {{per}\quad {ml}} \right)}} \\{= {130.29\quad {µm}^{2} \times 4.29 \times 10^{9}\quad {per}\quad {ml}}} \\{= {0.56\quad m^{2}\quad {ml}^{- 1}}}\end{matrix}$

[0171] The above parameters are calculated and displayed along with thecharacteristic curve of osmolality against percentage change in voltagein FIG. 10b.

[0172]FIG. 12 illustrates the three-dimensional frequency distributionof a sample from a patient having an HbCC disease. As shown, the plot isgrossly abnormal.

1. A method in which a sample of cells suspended in a liquid medium,wherein the cells have at least one measurable property distinct fromthat of the liquid medium, is subjected to analysis by a methodincluding the steps of: (a) passing a first aliquot of the sample cellsuspension through a sensor, (b) measuring said at least one property ofthe cell suspension, (c) recording the measurement of said property forthe first aliquot of cells, (d) subjecting the first or at least oneother aliquot of the sample cell suspension to an alteration in at leastone parameter of the cell environment which has the potential to alterthe shape of the cells to a known or identifiable extent to create analtered cell suspension, (e) passing said altered cell suspensionthrough a sensor, (f) measuring said at least one property of thealtered cell suspension, (g) recording the measurement of said at leastone property for said altered suspension, (h) comparing the data fromsteps (c) and (g) and determining a shape compensation factor to beapplied to the measurement of said at least one property of the firstaliquot of cells in step (c) in the calculation of a cell parameter totake account of a variation in shape between the first aliquot of cellsin step (c) and said altered cell suspension in step (g).
 2. A methodaccording to claim 1, in which the property of the cells which differsfrom the liquid medium is one which is related to the volume of thecell.
 3. A method according to claim 1 or 2, in which the cell propertyis electrical resistance or impedance.
 4. A method according to anypreceding claim, in which the environmental parameter change is analteration in osmolality.
 5. A method according to any preceding claimin which the environmental parameter change is an alteration inosmolality, and wherein the shape compensation factor determined in step(g) is given by the expression:${f\left( K_{shape} \right)} = {1 + {\left\lbrack \frac{\left( {{SR1} - {SR2}} \right)}{({SR1})} \right\rbrack \times K_{a}}}$

where K shape is the shape compensation factor, SR1 is a sensor readingat a known or identifiable shape, SR2 is a sensor reading at anosmolality of interest, and Ka is an apparatus dependent constant.
 6. Amethod according to claim 5, in which the sensor reading is one ofvoltage amplitude.
 7. A method according to claim 6, in which the volumeof a cell at an osmolality of interest is determined from theexpression: Cell Volume=SR n×K _(volts) ×K _(shape) where SRn is asensor reading at the osmolality of interest, K_(volts) is apredetermined voltage to cell volume conversion factor, and K_(shape) isthe shape compensation factor determined for the sensor reading SRn. 8.A method according to any of claims 5 to 7, in which said known oridentifiable shape is a spherical shape.
 9. A method according to anypreceding claim, in which the sample is fed continuously into asolution, the osmolality of which is changed continuously to produce acontinuous gradient of aliquots for passage through the sensing zone.10. A method according to claim 9, in which identical portions of thesample under test are subjected to solutions of each osmolalitythroughout the range under test after substantially the same time fromimposition of the environmental parameter change to the time of passagethrough the sensing zone.
 11. A method according to any preceding claim,in which said at least one property of the altered cell suspension ismeasured as each of a number of cells passes through the sensor, themeasurement of said at least one property for the altered cellsuspension is recorded on a cell-by-cell basis, and the data from steps(c) and (g) is compared as a functions of the extent of said alterationof said parameter of the cell environment and frequency distribution ofsaid at least one property.
 12. A method according to claim 11, furthercomprising the step of displaying the results of the analysis in theform of a three dimensional plot.
 13. A method according to anypreceding claim, in which the second aliquot of the sample cellsuspension is subjected to an alteration in at least one parameter ofthe cell environment which has the potential to induce a flow of fluidacross the cell membranes and thereby alter the said at least oneproperty of the cells, wherein data from steps (c) and (g) is comparedas a function of the extent of said alteration of said parameter of thecell environment and change in the recorded measurements of said atleast one property to determine a measure of cell permeability of thesample.
 14. An apparatus for testing a sample cell suspension in aliquid medium in accordance with the method any preceding claim,comprising data processing means programmed to compare data from saidsteps (c) and (g) to determine a shape compensation factor to be appliedto the measurement of said at least one property of the first aliquot ofcells in the calculation of a cell parameter to take account of avariation in shape between the first aliquot of cells and said alteredcell suspension.
 15. An apparatus according to claim 14, in which thedata processing means is programmed to compare data from steps (c) and(g) on a cell-by-cell basis as a function of the extent of saidalteration of said parameter of the cell environment and frequencydistribution of said at least one property.
 16. An apparatus accordingto claim 14 or 15, in which the data processing means comprises theinternal microprocessor of a personal computer.
 17. A method in which asample of cells suspended in a liquid medium, wherein the cells have atleast one measurable property distinct from that of the liquid medium,is subjected to analysis by a method including the steps: (a) passing afirst aliquot of the sample cell suspension through a sensor, (b)measuring said at least one property of the cell suspension as each of anumber of cells of the first aliquot passes through the sensor, (c)recording the measurement of said property for the first aliquot ofcells on a cell-by-cell basis, (d) subjecting the first or at least oneother aliquot of the sample cell suspension to an alteration in at leastone parameter of the cell environment which has the potential to alterthe said at least one property of the cells to create an altered cellsuspension, (e) passing said altered cell suspension through a sensor,(f) measuring said at least one property of the altered cell suspensionat each of a number of cells of the altered cell suspension passesthrough the sensor. (g) recording the measurement of said at least oneproperty for the altered cell suspension on a cell-by-cell basis, (h)comparing the data from steps (c) and (g) as a function of the extent ofsaid alteration of said parameter of the cell environment and frequencydistribution of said at least one property.
 18. A method according toclaim 17, in which the environmental parameter change is an alterationin osmolality.
 19. A method according to claim 17 or 18, in which thesample is fed continuously into a solution, the osmolality of which ischanged continuously to produce a continuous gradient of aliquots forpassage through the sensing zone.
 20. A method according to any ofclaims 17 to 19, further comprising the step of displaying the resultsof the analysis in the form of a three dimensional plot.